Douglass J. Forbes

Nuclear import can be separated into distinct steps in vitro: Nuclear pore binding and translocation

Large nuclear proteins must possess a signal sequence to pass through the nuclear pores. Using an in vitro system, we have been able experimentally to dissect nuclear protein transport into two distinct steps: binding and translocation. In the absence of ATP, we observe a binding of nuclear proteins to the pore that is signal sequence-dependent. Translocation through the pore, on the other hand, strictly requires ATP. These steps, visualized in the fluorescence and electron microscopes, were observed both with a natural nuclear protein, nucleoplasmin, and a synthetic nuclear protein, composed of the signal sequence of SV40 T antigen coupled to HSA. When a mutant signal sequence was coupled to HSA, neither transport nor binding were observed, indicating that both result from the presence of a functional signal sequence. An inhibitor of transport, the lectin WGA, also arrested nuclear proteins in a bound state at the cytoplasmic face of the pore. Therefore, only the translocation step is sensitive to the inhibitor WGA, which is known to bind specifically to proteins of the nuclear pore.

Mitotic repression of the transcriptional machinery

Nuclear RNA transcription is silenced when eukaryotic cells enter mitosis. Until recently, this repression was thought to derive solely from the condensation of interphase chromatin into mitotic chromosomes. Recent studies, however, have shown that changes in chromatin structure and occupancy of promoter elements by both general and gene-specific transcription factors also play a role in transcriptional silencing. In addition, studies with simplified systems reveal that reversible phosphorylation of the basal transcriptional machinery represses transcription at mitosis.

Nuclear Export Receptors: From Importin to Exportin

In a literal sense, export receptors may next interact with proteins of the nuclear pore (for example,Powers et al. 1997xPowers, M.A., Forbes, D.J., Dahlberg, J.E., and Lund, E. J. Cell Biol. 1997; 136: 241–250Crossref | PubMed | Scopus (152)See all ReferencesPowers et al. 1997; see alsoDoye and Hurt 1997xDoye, V. and Hurt, E. Curr. Opin. Cell Biol. 1997; 9: 401–411Crossref | PubMed | Scopus (192)See all ReferencesDoye and Hurt 1997). Another option is that other soluble factors may be required to bind exportins to the pore. Rip/Rab may belong to an as-yet-uncharacterized class of proteins that act as “itinerant” nucleoporins, soluble at some times and docked at the pore at others. By forming a Rip/exportin 1/Rev-NES complex, such proteins could act to dock the exportins at the pore. Indeed, evidence for such a complex was recently found (Neville et al. 1997xNeville, M., Lee, L., Stutz, F., Davis, L., and Rosbash, M. Curr. Biol., in press. 1997; See all ReferencesNeville et al. 1997).In the broader sense of where export receptors go next, questions for the future include: Do more exportins exist? If so, are they importin β–related or different? Are there specialized exportins that have distinct cargoes? Does signal transduction alter the exportins used? With exportins now in hand, meaningful experiments on the mechanism of export will soon follow.

Nuclear pores and nuclear assembly

Communication between the nucleus and cytoplasm occurs through large macromolecular structures, the nuclear pores. Quantitative scanning transmission electron microscopy has estimated the mass of a nuclear pore to be 60 million Daltons in yeast and 120 million Daltons in vertebrates. The past two years were noteworthy in that they saw: 1) the purification of both the yeast and vertebrate nuclear pores, 2) the initial description of routes through the pore for specific transport receptors, 3) glimpses of intranuclear organization imposed by the nuclear pores and envelope and 4) the revelation of new and pivotal roles for the small GTPase Ran not only in nuclear import but in spindle assembly and nuclear membrane fusion.

Removal of a Single Pore Subcomplex Results in Vertebrate Nuclei Devoid of Nuclear Pores

The vertebrate nuclear pore complex, 30 times the size of a ribosome, assembles from a library of soluble subunits and two membrane proteins. Using immunodepletion of Xenopus nuclear reconstitution extracts, it has previously been possible to assemble nuclei lacking pore subunits tied to protein import, export, or mRNA export. However, these altered pores all still possessed the bulk of pore structure. Here, we immunodeplete a single subunit, the Nup107-160 complex, using antibodies to Nup85 and Nup133, two of its components. The resulting reconstituted nuclei are severely defective for NLS import and DNA replication. Strikingly, they show a profound defect for every tested nucleoporin. Even the integral membrane proteins POM121 and gp210 are absent or unorganized. Scanning electron microscopy reveals pore-free nuclei, while addback of the Nup107-160 complex restores functional pores. We conclude that the Nup107-160 complex is a pivotal determinant for vertebrate nuclear pore complex assembly.

Reconstitution of biochemically altered nuclear pores: Transport can be eliminated and restored

Biochemically altered nuclear pores specifically lacking the N-acetylglucosamine-bearing pore proteins were constructed in a nuclear assembly extract in order to assign function to these proteins. The depleted pores do not bind nuclear signal sequences or actively import nuclear proteins, but they are functional for diffusion. These defects can be fully repaired by assembly with readded Xenopus pore glycoproteins. Strikingly, isolated rat pore glycoproteins also restore transport. Electron microscopy reveals that depleted pores have largely normal morphology. Thus, the pore glycoproteins are not required for assembly of the nuclear envelope, the major structures of the pore, or a pore diffusional channel. Instead, they are essential for active protein import and, unexpectedly, for construction of the part of the pore necessary for signal sequence recognition.

ELYS is a dual nucleoporin/kinetochore protein required for nuclear pore assembly and proper cell division

Nuclear pores span the nuclear envelope and act as gated aqueous channels to regulate the transport of macromolecules between the nucleus and cytoplasm, from individual proteins and RNAs to entire viral genomes. By far the largest subunit of the nuclear pore is the Nup107–160 complex, which consists of nine proteins and is critical for nuclear pore assembly. At mitosis, the Nup107–160 complex localizes to kinetochores, suggesting that it may also function in chromosome segregation. To investigate the dual roles of the Nup107–160 complex at the pore and during mitosis, we set out to identify binding partners by immunoprecipitation from both interphase and mitotic Xenopus egg extracts and mass spectrometry. ELYS, a putative transcription factor, was discovered to copurify with the Nup107–160 complex in Xenopus interphase extracts, Xenopus mitotic extracts, and human cell extracts. Indeed, a large fraction of ELYS localizes to the nuclear pore complexes of HeLa cells. Importantly, depletion of ELYS by RNAi leads to severe disruption of nuclear pores in the nuclear envelope, whereas lamin, Ran, and tubulin staining appear normal. At mitosis, ELYS targets to kinetochores, and RNAi depletion from HeLa cells leads to an increase in cytokinesis defects. Thus, we have identified an unexpected member of the nuclear pore and kinetochore that functions in both pore assembly at the nucleus and faithful cell division.

Novel vertebrate nucleoporins Nup133 and Nup160 play a role in mRNA export.

RNA undergoing nuclear export first encounters the basket of the nuclear pore. Two basket proteins, Nup98 and Nup153, are essential for mRNA export, but their molecular partners within the pore are largely unknown. Because the mechanism of RNA export will be in question as long as significant vertebrate pore proteins remain undiscovered, we set out to find their partners. Fragments of Nup98 and Nup153 were used for pulldown experiments from Xenopus egg extracts, which contain abundant disassembled nuclear pores. Strikingly, Nup98 and Nup153 each bound the same four large proteins. Purification and sequence analysis revealed that two are the known vertebrate nucleoporins, Nup96 and Nup107, whereas two mapped to ORFs of unknown function. The genes encoding the novel proteins were cloned, and antibodies were produced. Immunofluorescence reveals them to be new nucleoporins, designated Nup160 and Nup133, which are accessible on the basket side of the pore. Nucleoporins Nup160, Nup133, Nup107, and Nup96 exist as a complex in Xenopus egg extracts and in assembled pores, now termed the Nup160 complex. Sec13 is prominent in Nup98 and Nup153 pulldowns, and we find it to be a member of the Nup160 complex. We have mapped the sites that are required for binding the Nup160 subcomplex, and have found that in Nup98, the binding site is used to tether Nup98 to the nucleus; in Nup153, the binding site targets Nup153 to the nuclear pore. With transfection and in vivo transport assays, we find that specific Nup160 and Nup133 fragments block poly[A]+ RNA export, but not protein import or export. These results demonstrate that two novel vertebrate nucleoporins, Nup160 and Nup133, not only interact with Nup98 and Nup153, but themselves play a role in mRNA export.

Global histone acetylation induces functional genomic reorganization at mammalian nuclear pore complexes

The nuclear localization of genes is intimately tied to their transcriptional status in Saccharomyces cerevisiae, with populations of both active and silent genes interacting with components of the nuclear envelope. We investigated the relationship between the mammalian nuclear pore and the human genome by generating high-resolution, chromosome-wide binding maps of human nucleoporin 93 (Nup93) in the presence and absence of a potent histone deacetylase inhibitor (HDACI). Here, we report extensive genomic reorganization with respect to the nuclear pore following HDACI treatment, including the recruitment of promoter regions, euchromatin-rich domains, and differentially expressed genes. In addition to biochemical mapping, we visually demonstrate the physical relocalization of several genomic loci with respect to the nuclear periphery. Our studies show that inhibiting HDACs leads to significant changes in genomic organization, recruiting regions of transcriptional regulation to mammalian nuclear pore complexes.

Separate nuclear import pathways converge on the nucleoporin Nup153 and can be dissected with dominant-negative inhibitors

BACKGROUND Proteins generally enter or exit the nucleus as cargo of one of a small family of import and export receptors. These receptors bear distant homology to importin beta, a subunit of the receptor for proteins with classical nuclear localisation sequences (NLSs). To understand the mechanism of nuclear transport, the next question involves identifying the nuclear pore proteins that interact with the different transport receptors as they dock at the pore and translocate through it. RESULTS Two pathways of nuclear import were found to intersect at a single nucleoporin, Nup153, localized on the intranuclear side of the nuclear pore. Nup153 contains separate binding sites for importin alpha/beta, which mediates classical NLS import, and for transportin, which mediates import of different nuclear proteins. Strikingly, a Nup153 fragment containing the importin beta binding site acted as a dominant-negative inhibitor of NLS import, with no effect on transportin-mediated import. Conversely, a Nup153 fragment containing the transportin binding site acted as a strong dominant-negative inhibitor of transportin import, with no effect on classical NLS import. The interaction of transportin with Nup153 could be disrupted by a non-hydrolyzable form of GTP or by a GTPase-deficient mutant of Ran, and was not observed if transportin carried cargo. Neither Nup153 fragment affected binding of the export receptor Crm1 at the nuclear rim. CONCLUSIONS Two nuclear import pathways, mediated by importin beta and transportin, converge on a single nucleoporin, Nup153. Dominant-negative fragments of Nup153 can now be used to distinguish different nuclear import pathways and, potentially, to dissect nuclear export.